This project intends to elucidate some of the synthetic mechanisms involved in E. coli DNA replication by examining DNA intermediates and whole chromosomes in mutants which are defective in specific components of the replication machinery. A major goal of this project is to define the strand specificity of the two discrete size classes of small DNA intermediates generated during discontinuous DNA synthesis. A companion goal is to determine the gene products involved in the synthesis and extension of each class. One approach utilizes T4 bacteriphage as a probe to determine the function of one of these gene products. A second project is to isolate and characterize, both genetically and biochemically, a set of RNaseH mutants to determine if this enzyme functions in normal DNA replication. The possible construction of a DNA replication protein interaction map based on the ability of double DNA-defective mutants to serve as hosts for lambda bacteriophage is proposed. Additionally, we intend to exploit our recent finding that one dnaE mutant shows two modes of linear DNA synthesis that are distinguishable by their drug sensitivities. This finding, if it truly reflects component parts of normal DNA replication, splits the process in a new and unexpected way. It is hoped and expected that this work will eventually make it possible to conduct similar studies in eukaryotes where it should be of manifold important in the study of differentiation and tumor cell growth.